The techniques of externally introducing any given gene into animal cells including human cells, and expressing the gene persistently in the cells are essential techniques in various industries utilizing biotechnologies. For example, industrial mass production of human monoclonal antibodies for use as pharmaceuticals requires the technique of persistently expressing genes of H-chain and L-chain of immunoglobulin at the same level. In gene therapy of congenital metabolic diseases, the technique of introducing a therapeutic gene into human tissue cells, and stably expressing the gene in the body for a long term is required.
1. Regarding Cell-Reprogramming Technology
Recently, a cell-reprogramming technology for producing useful cells by genetically converting the characteristic of normal tissue cells attracts attention. The technique of introducing genes into an animal cell and persistently expressing the genes is also a base technology essential for cell-reprogramming. For example, it is possible to prepare human induced pluripotent stem cells (iPS cells) by introducing a combination of four genes, OCT4, SOX2, KLF4, and c-MYC, or OCT4, SOX2, NANOG, and LIN28 into human normal fibroblasts, and expressing the genes persistently for 21 days (Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2). Also, it is possible to prepare hepatic cells by introducing three genes, FOXA3, HNF1A, and HNF4A into human fibroblasts and expressing the gene persistently for 14 days. (Non-Patent Document 3) It is also reported that a dopaminergic neuron can be prepared by introducing five genes, ASCL1, BRN2, MYT1L, LMX1A, and FOXA2 into human fibroblasts, and expressing the gene persistently for 24 days (Non-Patent Document 4). Thus, in various cell-reprogramming, there is a need for a technique capable of simultaneously introducing and expressing plural genes into a cell, and keeping the expression for a period required for reprogramming.
It is known that cell-reprogramming can be induced in vivo. For example, it has been reported that when three genes, GATA4, MEF2C, and TBX5, or four genes, GATA4, HAND2, MEF2C, and TBX5 are administered to an infarcted site in a mouse myocardial infarction model, infiltrated fibroblasts transdifferentiate into cardiomyocytes (Non-Patent Document 5, and Non-Patent Document 6). Therefore, the cell-reprogramming technology is expected to become the basis of regenerative medicine for myocardial infarction, spinal cord injury and the like in future.
2. Improvement in Cell-Reprogramming Efficiency
Assuming that vitro cell-reprogramming is used for medicine, it is desired that the material cells can be collected from a human body without invasion, and can be collected in the condition that they are not contaminated with microorganisms outside the living body. Cells that satisfy these requirements are almost limited to mononuclear cells in peripheral blood, and a gene introduction vector adapted to these cells is desired.
In general, the efficiency with which animal cells are reprogrammed by externally introduced genes is very low, however, the efficiency can be raised by carrying all the genes on one vector, and introducing the genes into cells at once (Patent Document 3, Patent Document 4, Non-Patent Document 7, and Non-Patent Document 8).
Also it is known that the efficiency is raised by increasing the number of genes used in cell-reprogramming. For example, in the technique of converting mouse fibroblasts to induced pluripotent stem cells (iPS cells), it is known that the efficiency of conversion rises five times by using a total of six genes by adding two genes, BRG1 and BAF155, to four genes, OCT4, SOX2, KLF4, and c-MYC (Non-Patent Document 9). Also, in the technique of reprogramming human fibroblasts into motor nerves, it is known that the efficiency of reprogramming rises 100 times by using a total of seven genes by adding three genes, HB9, ISL1, and NGN2 to four genes, LHX3, ASCL1, BRN2, and MYT1L (Non-Patent Document 10).
When the number of genes used in cell-reprogramming is increased, the size of genes that should be carried also increases. Illustrating preparation of iPS cells as an example, the total size of four genes, KLF4, OCT4, SOX2, and c-MYC is 4,774 base pairs, whereas the total size of genes after adding the two genes, BRG1 (5,040 base pairs) and BAF155 (3,318 base pairs) is 13,132 base pairs (Non-Patent Document 9). By adding CHD1 gene (5,133 base pairs) encoding a chromatin remodeling factor that is specifically expressed in embryonic stem cells and is expected to accelerate reprogramming of cells to iPS cells to four genes, KLF4, OCT4, SOX2, and c-MYC, the total size amounts to 9,907 base pairs, and by adding TET1 gene (6,429 base pairs) encoding a DNA demethylase to four genes, KLF4, OCT4, SOX2, and c-MYC, the total size amounts to 11,203 base pairs. The total size of the seven genes, LHX3, ASCL1, BRN2, MYT1L, HB9, ISL1, and NGN2 that are used in the technique of reprogramming human fibroblasts into motor nerves is 9,887 base pairs (Non-Patent Document 10).
Thus, in order to raise the efficiency of the cell-reprogramming, it is desired to use at least six or more genes, and a vector capable of carrying all of the genes at once is desired. Also, desired is a vector capable of expressing introduced exogenous genes even when the total size of the genes is 5,000 or more nucleotides, desirably 8,000 or more nucleotides.
The term vector used herein refers to a recombinant viral or non-viral nucleic acid-macromolecular substance complex that is composed of nucleic acid including exogenous genes, and is capable of introducing the nucleic acid into animal cells and expressing the genes.
It is known that in reprogramming of animal cells by expression of exogenous genes, the expression levels of the genes seriously affect the characteristics of the reprogrammed cells. For example, when four genes, OCT4, SOX2, KLF4, and c-MYC are expressed in mouse fibroblasts, it is known that iPS cells are generated when expression of the genes is weak, whereas cells having a totally different characteristic from iPS cells are generated when the expression of the genes is strong (Non-Patent Document 11). Thus, for the technique of reprogramming animal cells including human cells by expressing externally introduced genes, there is a need for a vector capable of setting the expression of the genes at an optimum level depending on the purpose.
3. Removal of Genes for Reprogramming
Further in order to make the reprogrammed cells prepared by externally introducing genes completely exert their function, it is necessary to completely remove the reprogramming genes from the cells. Also, when the prepared human cells are used as a material for regenerative medicine, it is necessary to completely remove the genes from the cells for ensuring the safety. For example, in induced pluripotent stem cells (iPS cells) prepared by using four genes, OCT4, SOX2, KLF4, and c-MYC, the pluripotency cannot be functional in the condition that these four genes are expressed, and hence, it is necessary to at least completely suppress the expression of these genes, or preferably completely remove these genes from the cells (Patent Document 1, Patent Document 2, Patent Document 3, Non-Patent Document 1, Non-Patent Document 2 and Non-Patent Document 7). It is also known that if the c-MYC gene used in preparing the iPS cells is left in the iPS cells, the tissue cells that are prepared by differentiation of the iPS cells become tumorigenic with high frequency (Non-Patent Document 12). Therefore, it is necessary to completely remove the c-MYC gene from the iPS cells for ensuring the safety.
Thus, the gene expression technique required for cell-reprogramming needs to have the mutually contradictory characteristics: persistent expression of genes at an optimum levels is desired for achieving the reprogramming, while it can be removed easily and completely once the reprogramming has completed.
4. Importance of Avoiding Activation of Innate Immune System
Most of the gene introduction/expression vectors that are currently used in animal cells are constructed using an animal viruses or plasmid DNAs prepared from microorganisms such as Escherichia coli as materials. However, an animal cell has an innate immune system that eliminates invading pathogens from outside (Non-Patent Document 13) and nucleic acids derived from viruses or microorganisms introduced from outside the cell are recognized as foreign substances, and the innate immune system is activated. When the degree activation of the innate immune system exceeds a certain level, cell death by the apoptosis is induced, and thus the efficiency of the reprogramming is deteriorated. When expression of interferon or inflammatory cytokines is induced by the activation of the innate immune system, inflammation is caused in the living body. In order to prevent such an undesired reaction, gene introduction/expression technique for cell-reprogramming is required to be capable of avoiding the activation of the innate immune system. This characteristic is important particularly in application to the regenerative medicine including in vivo cell-reprogramming as described in the above section 1.
5. Gene Introduction/Expression System for Ideal Cell-Reprogramming
From the foregoing investigation, there is a need for a gene introduction/expression technique satisfying the following at least five requirements as discussed in the above sections 1 to 4. so as to further ameliorate the cell-reprogramming technique for animal cells including human cells by using genes for industrial application.
(1) Capability of efficiently introducing exogenous genes into animal cells including human peripheral blood cells.
(2) Capability of persistently expressing the genes for any required period.
(3) Capability of avoiding the innate immune system possessed by cells in expression of the genes.
(4) Capability of expressing the genes even if the total length of the introduced exogenous genes is 5,000 or more nucleotides, desirably 8,000 or more nucleotides.
(5) Capability of simultaneously expressing at least six, desirably eight or more genes.
Also, it is greatly desired to further achieve the following points.
(6) Capability of regulating the expression levels of the genes. In particular, it is preferred that the expression level of each gene can be regulated individually when plural genes are introduced.
In applying gene-introduced cells, in particular, to transplantation techniques, the following point is also very important.
(7) Capability of removing the gene by a simple technique when the genes become unnecessary.
6. Technique of Introducing Plural Genes into Animal Cells
As a technique for introducing plural genes into animal cells including human cells from outside, and expressing the genes persistently in the cells, that has been reported to be applicable to cell-reprogramming, the following three techniques are known.
(1) Method of integrating the genes into nuclear genomic DNA.
(2) Method of carrying the genes on DNA capable of existing stably and independently from genomic DNA in a nucleus.
(3) Method of carrying the genes on RNA capable of existing in cytoplasm.
6-1. Method for Integrating Plural Genes into Nuclear Genomic DNA
In the method of integrating an exogenous gene into genomic DNA existing in a nucleus of cell by using a lentivirus vector (Non-Patent Document 8, and Non-Patent Document 14), transposon (Non-Patent Document 15, and Non-Patent Document 16), non-homologous recombination, homologous recombination or the like, the gene can exist stably as with the genomic DNA. However, once the gene is integrated into the genomic DNA, complicated operations such as introducing a sequence specific recombinase into cells are required for selectively removing the gene from the genomic DNA, and the gene cannot be removed securely from every cell (Non-Patent Document 15). Further, since integration of exogenous genes into genomic DNA requires DNA replication of host cells, the efficiency of gene introduction into cells having poor proliferation potency such as blood cells is very low. Further, the phenomenon of “insertional mutagenesis” that random integration of exogenous gene into genomic DNA causes disruption or abnormal activation of genes of the host is known, and hence, there exists a concern about the safety for medical application (Non-Patent Document 17).
6-2. Method for Carrying Plural Genes on a DNA that is Independent from Genomic DNA in Nucleus
As a method for carrying a exogenous gene on a DNA capable of existing stably in a nucleus of cell independently from genomic DNA, a method of using a circular DNA carrying a replication origin of genome of Epstein-Barr virus (Non-Patent Document 18), and a method of using an artificial chromosome containing a straight-chain giant DNA (Non-Patent Document 19) are known. These DNA molecules continue replication and are kept stably in nuclei of human cells, and the mechanism of this relies on the mechanism with which genomic DNA of host cells is replicated. Therefore, it is impossible to specifically inhibit only replication of the DNA carrying exogenous genes, and a technique for actively removing the DNA from cells has not been reported. Additionally, since division of a host cell is required for introducing the DNA molecule into a cell nucleus, the efficiency of gene introduction into cells having poor proliferation potency such as blood cells is very low. Further, since it is known that circular DNA in a cell nucleus is frequently incorporated into genomic DNA of the cell, the risk of insertional mutagenesis cannot be eliminated (Non-Patent Document 20).
6-3. Technique of Expressing Plural Genes from Single Vector DNA
Further, as described in the above sections 6-1. and 6-2., when DNA is used as a platform for gene expression, a technique of expressing plural genes from the single vector DNA is required. As such a technique, the following three methods are known: 1) a method of simply linking plural independent genes, and expressing the genes, 2) a method of expressing plural proteins from one messenger RNA (mRNA) by using an RNA structure called Internal Ribosome Entry Site (IRES), and 3) a method of expressing a fusion protein in which plural proteins are linked by 2A peptide.
It is known that in the method of linking plural independent genes, expression of genes is strongly suppressed due to mutual interference between genes (Non-Patent Document 21). In order to prevent this, it is necessary to insert a structure called an insulator between genes, and the insertion increases the size of the vector DNA, and complicates the structure of the vector DNA. While the case of expressing four genes installed on one DNA molecule has been reported in this method (Non-Patent Document 22), the case of simultaneously expressing five or more genes has not been reported.
In the method of expressing plural proteins from one messenger RNA (mRNA) by using IRES sequence, the translation efficiency of the protein positioned downstream IRES sequence is lower than, or sometimes 10% or less compared with the translation efficiency of the protein positioned upstream IRES sequence (Non-Patent Document 23). Additionally, since IRES sequence has a relatively large size and has a complicated structure, the method of using IRES sequence is mainly used for simultaneously expressing two proteins.
2A peptide has a structure consisting of 18 to 22 amino acid residues found in a positive-sense single-stranded RNA virus, and a fusion protein in which plural proteins are connected by 2A peptide are automatically cleaved at the time of synthesis and dissociated into the original plural proteins. In this technique, one proline residue is left at the N-terminus of each protein arising after cleavage, and 17 to 21 amino acid residues are left at the C-terminus, and these excess amino acid residues can influence on the function of the protein (Non-Patent Document 24). In addition, since the efficiency of cleavage at a 2A peptide site is largely influenced by the structure of the fusion protein, it is necessary to make trial and error requiring labors for preparing plural proteins efficiently (Non-Patent Document 25). In the method of connecting plural proteins by 2A peptide, the case of simultaneously expressing four proteins (Non-Patent Document 8) and the case of simultaneously expressing five proteins (Non-Patent Document 16) have been reported. Also the case of expressing four proteins by combining IRES sequence and 2A peptide has been reported (Non-Patent Document 4).
6-4. Method for Carrying Plural Genes on One RNA Existing in Cytoplasm
As described in the above sections 6-1. to 6-3., in the existing gene introduction/expression technique that uses DNA as a platform for gene expression, cell-reprogramming using four to five genes has been reported. However, as long as DNA is used as a platform for gene expression, it is not easy to simultaneously carry six or more genes and to achieve removal of the genes in a convenient way, and a technique satisfying at least all the five requirements required for ideal reprogramming shown in the above section 5. has not been reported.
Meanwhile, as a technique of cell-reprogramming by expressing plural genes that are externally introduced into animal cells including human cells using RNA as a platform, techniques of using a positive-sense RNA (Non-Patent Document 26, and Non-Patent Document 27), and techniques of using a negative-sense RNA (Patent Document 3, Patent Document 4, Patent Document 5, Patent Document 6, Non-Patent Document 7, Non-Patent Document 28, Non-Patent Document 29, and Non-Patent Document 30) have been reported.
6-4-1. Method of Using Positive-Sense RNA
As a technique of cell-reprogramming by using a positive-sense RNA capable of existing stably in cytoplasm, a technique of using a positive-sense single-stranded genomic RNA derived from Venezuelan equine encephalomyelitis virus (VEEV) (Non-Patent Document 26) has been reported. In this technique, expression of four proteins is realized by replacing a structural gene on 3′ side of genomic RNA of VEEV with genes encoding proteins that are linked by 2A peptide. This system induces extremely strong expression of interferon, and then combination with an anti-interferon stance (B18R protein derived from vaccinia virus) is necessarily required (Non-Patent Document 26). The efficiency of gene introduction depends on the gene introducing reagent to be applied, and cells capable of being reprogrammed is limited to adhesive cells such as fibroblasts. An RNA carrying exogenous genes is unstable, and disappears by removing B18R protein from the culture medium.
As a technique of cell reprogramming by using a positive-sense RNA, a technique using a chemically synthesized messenger RNA (mRNA) (Non-Patent Document 27) has been reported. In this prior art, after mixing plural mRNAs separately carrying up to five exogenous genes, the plural mRNAs are introduced into cells by using a gene introducing reagent. Since the expressions of the genes are transient, it is necessary to newly introduce the genes into the cells every day. Also, the gene introduction is limited to adhesive cells such as fibroblasts. Also in this technique, since the innate immune system is activated strongly, it is necessary to combine an anti-interferon substance (B18R protein derived from vaccinia virus) (Non-Patent Document 27).
6-4-2. Method of Using Negative-Sense RNA
As a technique of cell-reprogramming using negative-sense RNAs, a method of using mixed vectors separately carrying an exogenous gene on a wild-type strain of Sendai virus which is one species of paramyxoviruses (Patent Document 5, Non-Patent Document 28, and Non-Patent Document 29), and a method of using a vector carrying three genes simultaneously (Patent Document 6, and Non-Patent Document 30) have been reported as prior arts. In these gene expression systems using negative-sense RNA(s), autonomous replication ability of the wild-type virus is attenuated by deleting F gene, and exogenous genes are installed respectively as single gene expression cassettes. Although activation of the innate immune system was not mentioned, the vectors are expected to have ability to activate the innate immune system correspondingly because it has been known that Sendai virus which is a material has strong interferon inducibility (Non-Patent Document 31). Also it has been reported that the vector can be removed by introducing a temperature sensitive mutation into genome of the wild-type virus, and thus increasing the cultivation temperature (Patent Document 6, Non-Patent Document 29, and Non-Patent Document 30). The size of gene that can be expressed by a vector based on wild-type Sendai virus has been reported to be from 3078 base pairs (beta galactosidase from Escherichia coli) (Non-Patent Document 32) to 3450 base pairs (sum of three genes, KLF4, OCT4, and SOX2) (Patent Document 6, Non-Patent Document 30).
As technique of reprogramming cells by using a negative sense RNA, a technique based on a mutant Sendai virus capably of persistent infection has been reported (Patent Document 3, Patent Document 4, and Non-Patent Document 7). In this technique, plural point mutations responsible for long-term persistence are identified in genome of the virus which is a material of the vector, and it is indicated that these mutations are involved in avoidance of activation of the innate immune system (deterioration in interferon expression). Also by deleting three genes from virus genome, and carrying new genes, it is possible to express four exogenous genes simultaneously. Further, it has been reported that vectors are actively removed from cells by suppressing expression of L gene that encodes an RNA-dependent RNA polymerase by short interfering RNA (siRNA). It has been reported that the size of gene that can be expressed with the use of a vector based on a mutant Sendai virus capable of persistent infection is 4774 base pairs (sum of four genes, KLF4, OCT4, SOX2, and c-MYC) (Patent Document 3, Patent Document 4, and Non-Patent Document 7).
7. Future Challenge in Plural Gene Introducing Techniques
In existent gene introduction/expression techniques using RNA as a platform for gene expression as described in the above section 6-4., cell-reprogramming using four to five genes has been reported. Among these techniques, a defective and persistent expression Sendai virus vector described in the above section 6-4-2. has the most excellent characteristic, however, the number of genes that can be installed on the vector has been reported to be at most four. In the technique using an RNA virus as a material, it is difficult to alter the level of gene expression.
As shown in the above section 6., the technique of externally introducing plural genes into animal cells including human cells and persistently expressing the genes in the cells has been variously modified toward optimization for cell-reprogramming that converts the characteristics of normal tissue cells using genes, and produces useful cells. However, a technique satisfying all the five requirements required for ideal reprogramming shown in the above section 5. has not been reported heretofore.